key: cord-0739323-z10evb1f
authors: Neumann, J.; Prezzemolo, T.; Vanderbeke, L.; Roca, C. P.; Gerbaux, M.; Janssens, S.; Willemsen, M.; Burton, O.; Van Mol, P.; Van Herck, Y.; CONTAGIOUS co-authors,; Wauters, J.; Wauters, E.; Liston, A.; Humblet-Baron, S.
title: An open resource for T cell phenotype changes in COVID-19 identifies IL-10-producing regulatory T cells as characteristic of severe cases
date: 2020-06-02
journal: nan
DOI: 10.1101/2020.05.31.20112979
sha: 1a903c5a8d5db2d8c76efdbf73763b8cb779c533
doc_id: 739323
cord_uid: z10evb1f

The pandemic spread of the novel coronavirus SARS-CoV-2 is due, in part, to the immunological properties of the host-viral interaction. The clinical presentation varies greatly from individual to individual, with asymptomatic carriers, mild to moderate-presenting patients and severely affected patients. Variation in immune response to SARS-CoV-2 may underlie this clinical variation. Using a high dimensional systems immunology platform we have analysed the peripheral blood compartment of 6 healthy individuals, 23 mild-to-moderate COVID-19 patients and 20 severe COVID-19 patients. We identify distinct immunological signatures in the peripheral blood of the mild-to-moderate and severe COVID-19 patients, including T cell lymphopenia, more consistent with peripheral hypo- than hyper-immune activation. Unique to the severe COVID-19 cases was a large increase in the proportion of IL-10-secreting regulatory T cells, a lineage known to possess anti-inflammatory properties in the lung. Annotated data is openly available (https://flowrepository.org/experiments/2713) with clinical correlates, as a systems immunology resource for the COVID-19 research community.

The novel coronavirus SARS-CoV-2 has infected millions of people worldwide, and impacted the society and the global economy in an unprecedented manner. The pandemic spread is due, in part, to the immunological properties of the host-viral interaction. Clinical presentation of SARS-CoV-2 infections (COVID-19) varies greatly from individual to individual, with asymptomatic carriers, mild-to-moderate-presenting patients and severely to critically affected patients 1, 2, 3, 4, 5, 6 . This clinical heterogeneity is responsible for the severity of the pandemic, with asymptomatic cases and long latency increasing the R0, and severe pathology at the other end of the clinical spectrum result in high mortality 1, 2, 3, 4, 5, 6, 7, 8 . Variation in immune response to SARS-CoV-2 may underlie this clinical disparity and unravelling the immunological features associated with progression into severe and life-threatening disease is highly needed in order to guide therapeutic decision making and biomarker discovery.

Immunological investigations during the SARS-CoV-2 pandemic have focused on identifying altered immune responses in COVID-19 patients. Innate immune response is an essential first line defense against viruses, including type I and III interferon (IFN). Recent studies have suggested that SARS-CoV-2 inhibits type I IFN production and signaling 9 , potentially explaining the long pre-symptomatic period and persistent viral load in many patients. Defects in NK cell function may also be present, with reports of higher expression of activation and exhaustion markers on NK cells, and impaired NK cytotoxicity and cytokine production 9,10 , and additional problems with innate immunity are likely to be identified. Other studies suggest a defect in the adaptive immune system. Lymphopenia is widely reported in COVID-19, and the severity of lymphopenia has been correlated with disease severity 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 .

Functional defects within T cells have been reported, with an increased number of non-functional CD4 + T cells and impaired T cell cytokine production 10, 11, 20 . In a series of 38 patients, an increase in naïve CD4 + was reported in COVID-19 patients, suggestive of impaired activation 21 .

This was confirmed in an independent study, but without significant changes between moderate and severe patients 22 , while another study reported no change 9 . Parallel findings were reported in broncho-alveolar lavage fluid of COVID-19 patients, with enrichment of naïve CD4 + T cells in severely affected patients 18 . Overall, it remains to be determined whether defects in innate or adaptive immunity or a synergistic effect of both underlie the unusually long infectious period of SARS-CoV-2.

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint Immune analysis is also contributing to an understanding of COVID-19 pathology. Parallels with other respiratory infections, such as influenza 23 , have led to the hypothesis that pathology is immune-mediated rather than due to direct viral induction, and the potential success of immunemodulating therapeutics in small-scale clinical trials provides preliminary support for this model 24, 25, 26, 27 . Neutrophils seem to be consistently elevated in severe patients and are associated with poor outcomes 10, 11, 12, 13, 14, 15, 16, 17, 22, 28 . Several studies have identified COVID-19 as a hyperinflammatory status, with a "cytokine storm" of pro-inflammatory cytokines 9, 11, 13, 14, 18, 29, 30, 31, 32, 33 .

IL-6, in particular, was consistently higher in severely affected patients compared to moderate and milder cases, suggesting it might be associated with disease severity 11, 13, 14, 18, 30, 32, 33 .

Inconsistent findings have been reported regarding the changes in myeloid subsets 9,12,34,35 , however, severe COVID-19 patients seem to have an increased number of inflammatory monocytes, producing higher levels of IL-6 and GM-CSF 12, 35 . Bronchoalveolar lavage fluid of severe COVID-19 cases also revealed high levels of monocytes and neutrophils as well as a proinflammatory environment 18,36 . Excessive T cell activation has also been suggested as a possible driver of disease, with increased expression of activation markers (such as HLA-DR, CD38, CD69, CD25, CD44, Ki-67, OX40 and CD137) by CD4 + and CD8 + T cells in severe patients 9, 11, 12, 20, 21, 33 . As with the cause of poor viral clearance, the cause of excessive immune pathology remains unclear.

The ambiguity of the COVID-19 immune profile is based, in part, on the recent origin of the pandemic, but also on the design of COVID-19 clinical trials, many of which have substandard design, lacking suitable controls or data transparency 37 . This probably is especially acute in studies on the phenotypic and functional changes of T cells, where the limited and sometimes contradictory reports may be attributed to the vagaries of cytometry. Altered gating hierarchy, use of alternative markers, different thresholds for defined expression -each can modify the outcome of analysis 38 . A potential solution is to provide open-access high dimensional data, allowing independent analysis with alternative strategies, while still preserving the rapid publication process required to deal with an evolving pandemic. As part of the CONTAGIOUS consortium, multi-omic approaches are being performed in a systematic and coordinated manner against a longitudinal COVID-19 cohort. Here we provide the first high dimensional flow cytometry analysis for the CONTAGIOUS cohort. The peripheral blood of 6 healthy individuals, . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint 23 mild-to-moderate COVID-19 patients and 20 severe COVID-19 patients, was assessed for changes to the T cell compartment, and identified a signature of IL-10-producing regulatory T cells in those patients with severe COVID-19. Annotated data is openly available (https://flowrepository.org/experiments/2713), allowing transparent and evolving data analysis.

As part of the CONTAGIOUS study into the immunome of COVID-19, patients were recruited through the University Hospital in Leuven, Belgium, beginning 27th March 2020. The dataset was generated on 6 healthy individuals, 23 mild-to-moderate COVID-19 patients (WHO score 3-4) and 20 severe COVID-19 patients (WHO score 5-7). Demographic data for the patients are summarized in Table 1 , with individual data on demographic and clinical values in

In order to determine the T cell phenotypes associated with clinical heterogeneity in COVID-19, we stimulated T cells ex vivo and used high parameter flow cytometry covering a comprehensive set of subset markers, activation markers and cytokines. First, we assessed the number of major leukocyte subsets in PBMCs, using key lineage markers (CD3, CD4, CD8, FOXP3, CD14, CD19). Cluster-based flow cytometry analysis, pooling all samples, separated leukocytes into populations that corresponded to CD4 + T cells, CD8 + T cells, CD19 + B cells, CD14 + monocytes and CD4 -CD8 -T cells ( Figure 1A,B, Supplementary Figure 1 ). Traditional gating was used to quantify these populations, and FOXP3 + regulatory T cells, in each sample. Despite the marked lymphopenia in COVID-19 patients (Figure 1C) , the relative proportion of these leukocyte populations remained unchanged with COVID-19 patients (Figure 1D ), other than a trend towards lower CD8 T cell numbers, indicating a non-specific mechanism of leukopenia. Subtle, but significant, differences were observed in the expression of lineage markers, with comparison of pooled mild-moderate and severe COVID-19 patient samples showing a more similar expression pattern than pooled healthy controls (Figure 1E) . At the individual level, healthy individuals were largely clustered together, while mild-moderate and severe COVID-19 patients exhibited similar cellular phenotypes (Figure 1F,G) . Together, these data indicate only subtle shifts in the balance of blood leukocyte populations with COVID-19, without any discrimination between mild-moderate and severe cases.

We next investigated the activation and polarization of conventional CD4 + T cells in COVID-19 patients. Using the expression of CD45RA, CCR7, 4-1BB, CD25, CTLA-4, HLA-DR, IFNγ, IL-. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. Using the same approach, we investigated the phenotype of FOXP3 + regulatory T cells. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint inflammatory TEM Tc1/Tc17 population (expressing both IFNγ and IL-17) was 13-fold more numerous in COVID-19 patients than in healthy controls, with 27/43 (63%) COVID-19 patients exhibiting numbers >2 standard deviations above the average of healthy individuals (Supplementary Resource 1). This population did not discriminate between mild-moderate and severe COVID-19 patients. At a global level, the cellular phenotype of CD8 + T cells was largely unchanged by COVID-19-status, using both pooled ( Figure 4D ) and individual (Figure 4E,F) analysis. Together this data suggests an ongoing CD8 T cell response in COVID-19 patients, with expansion of the potent Tc1/Tc17 TEM subset, however the comparative weakness of the response, and its failure to predict disease severity, argue against systemic CD8 T cell responses being a pathogenic driver.

As a resource to the COVID-19 research community we provide in Supplementary Resource 1 the quantification of leukocyte subsets at an individual level, paired with demographic and clinical correlates. We further advocate for independent analysis of this resource, with the raw data available from https://flowrepository.org/experiments/2713.

In this study we present an in-depth investigation of the T cell compartment of mild to severe COVID-19 patients, using a platform with the capacity to investigate both intracellular transcription factor expression and, in parallel, functional cytokine production on a single cell level. Strikingly, COVID-19 patients that required hospitalization due to their condition harbored a peripheral T cell landscape that did not differ substantially from the healthy control one regarding the viral response. This is in stark contrast to the normal phenotype arising with viral infections, which specifically trigger Th1/Tc1-driven responses, with increased secretion of IFNγ and cytotoxic capacity. This condition has been particularly well studied in the case of influenza which could be considered to date one of the most prevalent respiratory viral infections 40, 41, 42 .

However, patients infected with SARS-CoV-2 do not display this distinct Th1/Tc1 polarization.

While the mechanism for this polarization failure remains unknown, a potential explanation lies in the presence of a strong IL-6 environment 9,43,44 . IL-6 is known to affect the Th1/Tc1 response by direct inhibition of IFNγ gene expression 45, 46 . Accordingly, this observation would support ongoing clinical trials testing the efficacy of tocilizumab in COVID-19 patients.

Our results are in line with recent studies regarding the absence of pro-inflammatory cytokines in the T cell compartment 47 or even a decrease of CD4 + secreting IFNγ 11 . These contrast with data . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint identifying a Th1 signature in COVID-19 patients; the latter, however, being based on convalescent and non-hospitalized patients 48 . Intriguingly, IFNγ levels in COVID-19 patient serum have been reported to be slightly elevated compared to healthy controls 9 . If this is not due to differences in the patient cohort, the lack of a Tc1/Th1 phenotype would suggest a myeloid origin as the IFNγ source.

Our study was not able to unravel one of the most prominent features of COVID-19, the prevalent lymphopenia observed in most of the severely affected patients. In our study, both T and B lymphocyte compartments were affected equally, with a possible predominance for CD8 + T cells. Importantly, naïve T cells remained present in normal, or elevated, numbers, suggesting that T cell production/renewal was intact. Interestingly, this lymphocytopenia seems to be systemic, as previous studies in SARS-CoV-1 49 While the overall T cell compartment did not display major differences in comparison to healthy controls, there were two particular enriched T cell subsets in more severely affected patients that could reflect the inflammatory condition present in COVID-19 patients. The inflammatory subset Tc1/Tc17 represents highly activated T cells with high expression of PD-1 and HLA-DR in addition to its ability to secrete both IFNγ and IL-17 cytokines. The presence of this population may reflect a deviation from the normal Tc1 anti-viral response caused by the pro-inflammatory IL-6-enriched environment 58 . The contribution of this population to the inflammatory setting is, . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint however, debatable, as the increase in relative number is mitigated by the lymphopenia, and no differences were observed between the mild-moderate and severe COVID-19 patients.

The only peripheral biomarker that did delineate disease severity was the increase in IL-10producing regulatory T cells. Elevated IL-10 has been observed in the serum of COVID-19 patients before 11 , however the cellular source was not elucidated. Production of IL-10 is a hallmark of activated regulatory T cells that reside in tissues such as the lung. This population, normally rare in healthy individuals, rose up to ~10% of the regulatory T cell pool in severe COVID-19 patients. The murine analogue to this population has a potent ability to limit inflammation and tissue damage triggered by microbial and environmental interactions at mucosal surfaces 39, 59 . In the case of viral infections of the lung, IL-10 restrains the development of IL-17-producing cells that damage the tissue 60,61 , inhibits the innate inflammatory response to viral particles, and is likely beneficial in reducing the production of cytokines such as IL-6 that have been implicated in COVID-19 morbidity 62, 63 . Increase of this suppressive regulatory T cell subset could be a direct response to the progressing lung inflammation in COVID-19 patients, comprising a feedback inhibition circuit to prevent runaway inflammation and death 64 .

Potentially, elevated IL-10 could provide a blood-based biomarker for cases progressing to more severe lung damage. A more intriguing possibility is that individuals with higher IL-10producing regulatory T cells exhibit defective adaptive immunity. IL-10 + regulatory T cells are symptomatic of many unresolved viral infections and are associated with long-term persistence.

In respiratory infections, IL-10 potently suppresses anti-viral responses 59, 65 and weakens the immune reaction to superinfection with bacteria 66, 67 . Since secondary infection leading to pneumonia is a major cause of death in influenza, and perhaps for some COVID-19 patients as well 68, 69, 70 , excessive IL-10 production by regulatory T cells may be a key factor in COVID-19 outcomes. In principle, this truncated adaptive immune response could allow persistent infection and cause an over-reliance on innate responses, driving the pathological state. Under this latter model, early intervention (such as with IL-10 neutralization) could potentially restore appropriate adaptive immunity and quieten the excessively exuberant innate response in the tissue. However key replication, longitudinal and mechanistic studies would be first required, and IL-10 has proven stubbornly refractory to immune modulation in the past 71 .

Key limitations in our study are the study size and the cross-sectional nature. The data presented here rely on a small number of patients without longitudinal follow-up, and lacking asymptomatic patients to screen the full spectrum of the COVID-19 immune signature. Study expansion and follow-up are ongoing to correct these deficits, and identify key components of . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. 

Frozen PBMCs were thawed, plated and incubated for 4 hours with complete RPMI containing phorbol myristate acetate (PMA 50 ng/mL), ionomycin (500 ng/mL) and Brefeldin A (8 µg/mL; all Tocris Bioscience) at 37 °C with 5% CO2. Cells were then washed twice with PBS (Fisher . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. and severe COVID-19) were performed with tests on the cross-entropy distributions of the tSNE representations of each group. In brief, for the original and t-SNE space of each tSNE plot, a probability per data point was calculated following the same approach as in the tSNE algorithm.

From these point probabilities, the distribution of cross-entropy in the tSNE space relative to the original space was obtained for each group represented in the plot. All pair-wise comparisons . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint between groups were evaluated with Kolmogorov-Smirnov tests on the difference between the cross-entropy distributions. Resulting p-values were corrected with the Holm method.

Dendrograms were obtained from hierarchical clustering, using as distance the Kolmogorov-Smirnov statistic, that is, the L-infinity distance between the cross-entropy distributions (manuscript in preparation).

This work was supported by the VIB Grand Challenges Program, the KUL C1 program, the . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China

Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the

Clinical Characteristics of Coronavirus Disease 2019 in China

Baseline Characteristics and Outcomes of 1591 Patients Infected With SARS-CoV-2 Admitted to ICUs of the Lombardy Region

Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship

Universal Screening for SARS-CoV-2 in Women Admitted for Delivery

The reproductive number of COVID-19 is higher compared to SARS coronavirus

of Novel Coronavirus-Infected Pneumonia

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Clinical and immunological features of severe and moderate coronavirus disease 2019

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients

Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19). Front Immunol

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

Characteristics of Peripheral Lymphocyte Subset Alteration in COVID-19 Pneumonia

Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP)

Suppressed T cell-mediated immunity in patients with COVID-19: A clinical retrospective study in Wuhan

Study of the lymphocyte change between COVID-19 and non-COVID-19 pneumonia cases suggesting other factors besides uncontrolled inflammation contributed to multi-organ injury

COVID-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

Waste in covid-19 research

Computational flow cytometry: helping to make sense of high-dimensional immunology data

Regulatory T Cell-Derived Interleukin-10 Limits Inflammation at Environmental Interfaces

T Cell-Mediated Host Immune Defenses in the Lung

Dissecting the human immunologic memory for pathogens

Severe Influenza Is Characterized by Prolonged Immune Activation: Results From the SHIVERS Cohort Study

Longitudinal hematologic and immunologic variations associated with the progression of COVID-19 patients in China

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Inhibition of Th1 Differentiation by IL-6 Is Mediated by SOCS1

IL-6 promotes the differentiation of a subset of naive CD8+ T cells into IL-21-producing B helper CD8+ T cells

A single-cell atlas of the peripheral immune response to severe COVID-19

Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals

Pathology and Pathogenesis of Severe Acute Respiratory Syndrome

The Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Directly Decimates Human Spleens and Lymph Nodes

COVID-19 in patients with HIV: clinical case series

Outcomes among HIV-positive patients hospitalized with

COVID-19 in people living with human immunodeficiency virus: a case series of 33 patients

Immune Recovery after Allogeneic Hematopoietic Stem Cell Transplantation Following Flu-TBI versus TLI-ATG Conditioning

Coronavirus Disease Covid-19: Ebmt Recommendations Version 8

A possible role for B cells in COVID-19? Lesson from patients with agammaglobulinemia

Two X-linked agammaglobulinemia patients develop pneumonia as COVID-19 manifestation but recover

Tc17 cells are a proinflammatory, plastic lineage of pathogenic CD8+ T cells that induce GVHD without antileukemic effects

Viral Antigen Induces Differentiation of Foxp3 + Natural Regulatory T Cells in Influenza Virus-Infected Mice

Interleukin-10 Signaling in Regulatory T Cells Is Required for Suppression of Th17 Cell-Mediated Inflammation

Deficiency Unleashes an Influenza-Specific Th17 Response and Enhances Survival against High-Dose Challenge

Negative regulation of MyD88-dependent signaling by IL-10 in dendritic cells

Interleukin-6 in COVID-19: A Systematic Review and Meta-Analysis. medRxiv. 2020;2020.03.30

IL-10: A Multifunctional Cytokine in Viral Infections

A Detrimental Effect of Interleukin-10 on Protective Pulmonary Humoral Immunity during Primary Influenza A Virus Infection

Influenza A virus-induced release of interleukin-10 inhibits the anti-microbial activities of invariant natural killer T cells during invasive pneumococcal superinfection

IL-10 Is an Important Mediator of the Enhanced Susceptibility to Pneumococcal Pneumonia after Influenza Infection

Co-infections: potentially lethal and unexplored in COVID-19. The Lancet Microbe

Predominant Role of Bacterial Pneumonia as a Cause of Death in Pandemic Influenza: Implications for Pandemic Influenza Preparedness

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Interleukin-10 paradox: A potent immunoregulatory cytokine that has been difficult to harness for immunotherapy

Available from: https://onlinelibrary.wiley.com/doi/abs/10.1111/pai.13263 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.31.20112979 doi: medRxiv preprint